It is essential in all cells to regulate DNA replication precisely, as failures in this lead to cancer or developmental defects. Most, if not all, animals and plants have polyploid or polytene cells, the consequence of altered regulation of DNA replication to produce cells that can function as metabolic factories or serve as large support cells. In addition, some organisms over or underreplicate specific genomic regions to control gene expression. These developmental aspects of the regulation of DNA replication are the focus of this research. The approaches of genomics and cell biology will be used to understand how metazoan DNA replication is controlled during development, innovative methodologies for this biological problem. New examples of amplified and underreplicated genes have been identified in the fruit fly Drosophila melanogaster by a genomic microarray approach. The replicative properties and biological functions of these differentially replicated genes will be deciphered. The microarray methodology will be employed to determine how widely used differential replication is as a developmental strategy for gene expression in Drosophila. Initiation of DNA replication is controlled by a set of proteins conserved from yeast to humans, and a model system has been developed in Drosophila that permits analysis of these proteins and replication origins. Binding of the Origin Recognition Complex (ORC), the Cdtl/DUP initiator protein, and the MCM hexamer can be visualized directly in the ovarian follicle cells. Furthermore after replication initiation, the DUP and MCM) proteins can be seen moving with replication forks during elongation. This cell biological approach, combined with the ability to recover mutants and examine their defects in replication, provides a powerful means to delineate the regulatory circuitry for replication initiation. The role of the DUP protein and its inhibitor Geminin will be defined in cell cycles that produce polyploid or polytene cells. In the final aim, the mechanisms that inhibit mitosis to permit formation of polyploid or polytene chromosomes will be delineated, and it will be determined how proliferating cells are protected from becoming polyploid.